微流控单分子检测免疫分析技术的初步研究
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摘要
本论文分为三部分:Ⅰ.第一章,微流控单分子检测免疫分析条件的初步探讨。Ⅱ.第二章,微流控单分子检测免疫分析条件的优化及选择。Ⅲ.第三章,肿瘤相关抗原CA125的单分子检测的条件。
论文第一章对微流控技术,免疫分析技术,单分子检测进行了简单的介绍,引用文献60余篇。本章重点对微流控单分子检测免疫分析的条件进行了初步探讨。研究了PDMS膜的修饰方法,盖玻片硅烷化时间,驱动电压选取,缓冲溶液的选择等条件;初步选择微流控免疫分析的条件:用新生态氧气对PDMS膜修饰2d;盖玻片硅烷化时间24h;缓冲液冲洗条件:1000V驱动电压,输出时间20min。BSA封闭条件:1000V驱动电压,输出时间10s,静止时间50s,重复这一过程80次。抗体与目标抗原的最佳孵育条件:1000V驱动电压,输出时间10s,静止时间50s,重复这一过程80次。
论文第二章对微流控单分子检测免疫分析的条件在第一章的基础上做了进一步的研究。改变了储液池的直径及微通道的结构和尺寸,确定微通道的宽度150μm,深度～20μm,PDMS膜的厚度～5mm,储液池的直径2mm,通道末端21mm区域对应的载玻片进行硅烷化处理。在这一新制备的微流控芯片上,对缓冲液冲洗时间、BSA封闭时间、抗体与目标抗原孵育时间等条件做了进一步的研究。
论文第三章在前面两章所找到的最佳条件的基础上,在微流控芯片上采用双抗夹心法进行免疫反应,并结合全内反射荧光显微镜单分子成像检测,探讨了检测人卵巢癌抗原CA125的相关条件。
The thesis includes three parts:Ⅰ.The preliminary study of the conditions of microfluidic immunoassay by single molecules detection.ⅡThe optimization and choice of the conditions of microfluidic immunoassay by single molecules detection.Ⅲ.Detecting conditions of Cancer antigen 125 single molecules.
In chapter one of this dissertation,a brief review of microfluidic,immunoassay analysis and single molecules detection was made.In this part,60 references have been referred.Then the preliminary study of the conditions of microfluidic immunoassay by single molecules detection was made.Several parameters including modification of PDMS,silanization time,driving voltage,buffer system were optimized.The following conditions were suitable for microfluidic immunoassay: modified PDMS slab with nascent oxygen for 2 days;the glass coverslip was silanized with 3%GOPS for 24 h;buffer washing conditions:injection voltage,1000 V,injection time,20 min;blocking conditions:injection voltage,1000 V,injection time 10 s,quiet time 50 s,repeat this precess:80 times;incubation conditions: injection voltage,1000 V,injection time 10 s,quiet time 50 s,repeat this precess:80 times.
In chapter two of this dissertation,we made a further investigation about the microfluidic immnunoassay conditions on the base of chapter one.The diameter of the reservoirs,the fabrication and dimension of the microchannels were changed.All channels on the～5-mm thickness PDMS slab with a length of 4 cm and a width of 2 cm were～150μm wide and～20μm deep.On this chip we investigated the washing conditions,blocking time and incubation time etc..
In chapter three of this dissertation,we investigate the conditions of sandwich immunoassay with single molecule detection on the microfluidic chip.The following conditions were suitable for microfluidic immunoassay to detect CA 125:
论文第一章对微流控技术,免疫分析技术,单分子检测进行了简单的介绍,引用文献60余篇。本章重点对微流控单分子检测免疫分析的条件进行了初步探讨。研究了PDMS膜的修饰方法,盖玻片硅烷化时间,驱动电压选取,缓冲溶液的选择等条件;初步选择微流控免疫分析的条件:用新生态氧气对PDMS膜修饰2d;盖玻片硅烷化时间24h;缓冲液冲洗条件:1000V驱动电压,输出时间20min。BSA封闭条件:1000V驱动电压,输出时间10s,静止时间50s,重复这一过程80次。抗体与目标抗原的最佳孵育条件:1000V驱动电压,输出时间10s,静止时间50s,重复这一过程80次。
论文第二章对微流控单分子检测免疫分析的条件在第一章的基础上做了进一步的研究。改变了储液池的直径及微通道的结构和尺寸,确定微通道的宽度150μm,深度～20μm,PDMS膜的厚度～5mm,储液池的直径2mm,通道末端21mm区域对应的载玻片进行硅烷化处理。在这一新制备的微流控芯片上,对缓冲液冲洗时间、BSA封闭时间、抗体与目标抗原孵育时间等条件做了进一步的研究。
论文第三章在前面两章所找到的最佳条件的基础上,在微流控芯片上采用双抗夹心法进行免疫反应,并结合全内反射荧光显微镜单分子成像检测,探讨了检测人卵巢癌抗原CA125的相关条件。
The thesis includes three parts:Ⅰ.The preliminary study of the conditions of microfluidic immunoassay by single molecules detection.ⅡThe optimization and choice of the conditions of microfluidic immunoassay by single molecules detection.Ⅲ.Detecting conditions of Cancer antigen 125 single molecules.
In chapter one of this dissertation,a brief review of microfluidic,immunoassay analysis and single molecules detection was made.In this part,60 references have been referred.Then the preliminary study of the conditions of microfluidic immunoassay by single molecules detection was made.Several parameters including modification of PDMS,silanization time,driving voltage,buffer system were optimized.The following conditions were suitable for microfluidic immunoassay: modified PDMS slab with nascent oxygen for 2 days;the glass coverslip was silanized with 3%GOPS for 24 h;buffer washing conditions:injection voltage,1000 V,injection time,20 min;blocking conditions:injection voltage,1000 V,injection time 10 s,quiet time 50 s,repeat this precess:80 times;incubation conditions: injection voltage,1000 V,injection time 10 s,quiet time 50 s,repeat this precess:80 times.
In chapter two of this dissertation,we made a further investigation about the microfluidic immnunoassay conditions on the base of chapter one.The diameter of the reservoirs,the fabrication and dimension of the microchannels were changed.All channels on the～5-mm thickness PDMS slab with a length of 4 cm and a width of 2 cm were～150μm wide and～20μm deep.On this chip we investigated the washing conditions,blocking time and incubation time etc..
In chapter three of this dissertation,we investigate the conditions of sandwich immunoassay with single molecule detection on the microfluidic chip.The following conditions were suitable for microfluidic immunoassay to detect CA 125:
引文
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